SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists

F23
SCREENING OF BUPRENORPHINE IN URINE OF SUSPECTED ABUSERS BY ELISA
Asimah Hamzah*, P.C. Peh, C.M. Lim, C.P.Lui, PhD, Narcotics II Laboratory, Centre for Forensic
Science, Health Sciences Authority, 11 Outram Road, Singapore 169078
Introduction: Buprenorphine is a synthetic thebaine derivative that has both analgesic and opioid antagonist
properties. As an analgesic, it is 25 to 40 times more potent than morphine, and has a slower onset of pain
relief and longer duration of action. Hence it is used for the treatment of chronic pain, and in treatment of
heroin addiction as an alternative to methadone. In human, buprenorphine is metabolised primarily by N­
dealkylation to norbuprenorphine. Both the metabolite and the parent drug undergo extensive conjugation
to glucuronides that are excreted in urine. It is known that buprenorphine has been abused by heroin addict;
therefore there is a need for the laboratory to develop a screening method for the suspected abusers.
Aim: The purpose of this study is to evaluate the use of an Enzyme Linked Immunosorbent Assay (ELISA)
test kit in the screening of buprenorphine in urine to detect suspected abusers.
Method: The ELISA test kits were purchased from Neogen Corp. USA. The assay principle is based on the
competitive binding between the free drugs in the urine and the drug enzyme conjugates for antibodies,
which are coated onto the 96-well microplate. After incubating the urine and drug enzyme conjugate in the
wells for 45 min at room temperature, the wells were washed with buffer, and K·blue substrate was added
to each well. After a further 30 min of incubation, Red Stop solution was added to each well to stop the
reaction. Multiskan Ascent Microplate Photometer was used to measure the absorbance at wavelength of
650 nm. The color intensity of each well is inversely proportional to the concentration of the drug in the
urine. Confirmatory test, using Gas chromatography-mass spectrometry (GC·MS) was also carried out on
all the urine specimens to compare with the screening results.
Results: The linearity of the ELISA kit was found to be up to about 5 ng/ml of buprenorphine. The
coefficients of variation (CVs) of the within-day and between-day variations for buprenorphine ranged
from 2.0% to 6.7% and 8.3% to 19.1% respectively. Norbuprenorphine was found to cross react to certain
extend. No cross-reactivity was found with other common drugs of abuse such as morphine, codeine,
hydrocodone, hydromorphone, Il-nor-L'1 9 -THC-9-carboxylic acid, cocaine, benzoylecgonine, amphetamine,
methampethamine, N,a-Dimethyl-3,4-(methylenedioxy)phenethylamine (MDMA), a-Methyl-3,4­
(methylenedioxy)phenethylamine (MDA), N-Ethyl-a,methyl·3,4-(methylenedioxy)phenethYlamine
(MDEA), ketamine and norketamine. Out of the 3 urine specimens that were collected from suspected
abusers, 17 were found to be screened positive, using the cut-off at 1 ng/ml. The linearity range of
buprenorphine was found to be up to 50 ng/ml. The LOQ was found to be 0.25 nglml. The results from the
confirmatory test are also discussed.
Conclusions: The use of ELISA test kit was found to be a simple, rapid, specific and effective method for
the screening of buprenorphine in urine. It helps to minimize the cost of the GC-MS test in a mass
screening exercise.
Keywords: ELISA, Screening, Buprenorphine
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